The principle of countercurrent chromatography (CCC) is generally as follows: when a water-filled coil is held horizontal and slowly rotated around its own axis, any object therein either heavier or lighter than the water moves toward one end of the coil. This end is called the "head" and the other end the "tail" of the coil. When such a coil contains two immiscible solvents, slow rotation soon establishes a hydrodynamic equilibrium between the two solvent phases where the head side of the coil is occupied by nearly equal amounts of the two phases and any excess of either phase is found at the tail end of the coil. Under this hydrodynamic equilibrium condition, the coil can be eluted with one of the phases through the head end while retaining the other phase stationary in the coil. Consequently, solutes locally introduced at the head of the coil are subjected to an efficient partition process between the mobile and stationary phases and chromatographically separated according to their partition coefficients in the absence of solid supports. Eluate through the tail end of the coil is continuously monitored as to optical absorbance and then is fractionated, as in liquid chromatography.
Peak resolution produced by this CCC scheme is greatly influenced by volume of the stationary phase retained in the coil, i.e., the higher the retention level, the better is the result obtained. It has been observed that the retention of the stationary phase is quite sensitive to the orientation and rotational speed of the coil. In the coaxially rotated coil, slow rotation usually yields nearly 50% retention. Increase of the rotational speed of the coil radically changes the hydrodynamic equilibrium volume ratio of the two phases in the coil, and in some critical range, one of the phases almost entirely collects at the head end and the other phase at the tail end of the coil.
This equilibrium condition permits a high retention level of the stationary phase if the mobile phase is introduced in the proper direction. In the eccentric orientation of the coil, the centrifugal force field induced by the rotation tends to trap the heavier phase in the outer half and the lighter phase in the inner half of each helical turn, resulting in more or less even distribution of the two phases throughout the coil and, therefore, the retention of the stationary phase becomes rather insensitive to the rotational rate of the coil. Thus, eccentrically rotated coils usually retain the stationary phase in no more than 50% of the column space unless the axis of rotation is inclined relative to the horizontal plane.
In the prior art, countercurrent chromatography has been performed by the use of multiple units of individually wound coils eccentrically attached to and supported by a rotary shaft. Each coil unit was equipped with connectors at both ends, and a long column was made by connecting the desired number of these coil units in series. This column arrangement, however, usually retains the stationary phase at a level less than 50% of the total free space in the column. The retention of the stationary phase is an important parameter for determining the degree of peak resolution and the sample loading capacity.
More specifically, it is known that performance of CCC systems mainly depends upon the amount of the stationary phase retained in the column, which determines both the resolving power of the solute peaks and the sample loading capacity. Various CCC systems developed in the past (see Y. Ito, J. Biochem. Biophys. Met., 5 (l981) 105) are usually capable of yielding retention of the stationary phase of no more than 50% of the total column space. This maximum attainable retention level tends to fall rather sharply with the application of higher flow rates of the mobile phase, resulting in loss of peak resolution. Consequently, the applicable flow rate has become one of the major limiting factors in CCC, and the prior methods require relatively long separation times, ranging from overnight to several days to complete a sizable separation. There is a definite need for an improved CCC system which can perform using a relatively high feed rate of the sample solution and which requires a substantially shortened separation time (see Y. Ito, J. Chromatogr., 207 (1981) 161).